Accurate DNA replication is crucial for the maintenance of genomic stability and for the suppression of mutagenesis and carcinogenesis. DNA polymerase d (Pold) is a high fidelity polymerase that plays an indispensable role in replication from yeast to humans. Pold from the yeast S. cerevisiae is comprised of three subunits, Pol3, Pol31, and Pol32. Pol3 is the catalytic subunit of the holoenzyme, encoding both the polymerase and the 3' to 5' exonuclease proofreading functions. Mutations in either the polymerase or the exonuclease domain of Pol3 that lower the fidelity of Pold cause cancers in mice and humans. For example, mutations that map to Pol3 have been identified in cancer cell lines and in sporadic colon cancers. Here, we propose structural, biochemical, and genetic studies on yeast Pold that are crucial for understanding the action mechanisms of this high fidelity polymerase. We will: 1) Determine the crystal structures of Pol3 in the polymerizing and editing modes. The structures will provide a mechanistic understanding of the basis for the high selectivity of Pol3 for the correct nucleotide, and will yield insights into the conformational transitions that underlie its fidelity. 2) We will use the structural information to make mutations that a) alter the fidelity of DNA synthesis, and b) affect the transition to the editing mode. Together, these mutations will test specific hypotheses that are inferred from the structures to form the basis of nucleotide selection and to contribute to active site switching. We will examine the effects of these mutations on Pold function by both biochemical and genetic means. 3) To understand the contributions that the Pol31 and Pol32 subunits make to Pold structure and function, we will carry out biochemical and structural studies on the Pold holoenzyme. The effects of Pol31 and Pol32 on the DNA binding proficiency and on the processivity of DNA synthesis by Pold will be determined, and pre-steady state kinetic analyses will be carried out to identify the contributions that different steps of the polymerization reaction make to the high fidelity of Pold. In addition, we will determine the structure of Pold holoenzyme in ternary complex with DNA and dNTP. A comparison of the ternary structures of Pol3 and Pold holoenzyme will be invaluable for deciphering the contributions of Pol31 and Pol32 to Pold function. The combined structural, biochemical, and genetic approaches proposed here will be important for defining the action mechanisms of Pold's polymerizing and proofreading functions and for delineating the structural bases of its high fidelity. PUBLIC HEALTH RELEVANCE: The aim of this study is to uncover the mechanisms underlying the high fidelity of DNA polymerase 4 (Pol4), which is crucial for maintaining genomic stability and the suppression of carcinogenesis. Mutations that lower the fidelity of Pol4 cause cancers in mice and humans.